Abstract
BackgroundLactic acid bacteria (LAB) are important microorganisms in the food and beverage industry. Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In this present study, we aimed at constitutive expression of a mannanase from Bacillus licheniformis DSM13, which was subsequently displayed on the cell surface of Lactobacillus plantarum WCFS1, for use as whole-cell biocatalyst in oligosaccharide production.ResultsTwo strong constitutive promoters, Pgm and SlpA, from L. acidophilus NCFM and L. acidophilus ATCC4356, respectively, were used to replace the inducible promoter in the lactobacillal pSIP expression system for the construction of constitutive pSIP vectors. The mannanase-encoding gene (manB) was fused to the N-terminal lipoprotein anchor (Lp_1261) from L. plantarum and the resulting fusion protein was cloned into constitutive pSIP vectors and expressed in L. plantarum WCFS1. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The mannanase activity and the reusability of the constructed L. plantarum displaying cells were evaluated. The highest mannanase activities on the surface of L. plantarum cells obtained under the control of the Pgm and SlpA promoters were 1200 and 3500 U/g dry cell weight, respectively, which were 2.6- and 7.8-fold higher compared to the activity obtained from inducible pSIP anchoring vectors. Surface-displayed mannanase was shown to be able to degrade galactomannan into manno-oligosaccharides (MOS).ConclusionThis work demonstrated successful displaying of ManB on the cell surface of L. plantarum WCFS1 using constitutive promoter-based anchoring vectors for use in the production of manno-oligosaccharides, which are potentially prebiotic compounds with health-promoting effects. Our approach, where the enzyme of interest is displayed on the cell surface of a food-grade organism with the use of strong constitutive promoters, which continuously drive synthesis of the recombinant protein without the need to add an inducer or change the growth conditions of the host strain, should result in the availability of safe, stable food-grade biocatalysts.
Highlights
Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry
We investigated two constitutive promoters, Pgm, the promoter of a phosphoglycerate mutase from L. acidophilus NCFM, and SlpA, the promoter of a well-characterized S-layer protein SlpA of L. acidophilus ATCC 4356, for the expression, secretion and display of a β-mannanase from Bacillus licheniformis DSM13 in L. plantarum WCFS1 using a lipoprotein anchor Lp_1261 from L. plantarum [25, 26]
Constitutive expression of ManB in L. plantarum Inducible lactobacillal expression vector pSIP409 was selected as a starting point for the construction of the vectors for constitutive expression, secretion and anchoring of mannanase (ManB) to cell membrane of L. plantarum
Summary
Lactic acid bacteria (LAB) are important microorganisms in the food and beverage industry Due to their food-grade status and probiotic characteristics, several LAB are considered as safe and effective cell-factories for food-application purposes. In the so-called genetic immobilization, proteins of interest, which are fused with the anchoring motifs, are synthesized and subsequently anchored on the bacterial cell surface, the immobilized enzymes can be obtained from the cultivations, using the bacterial biomass as the immobilization matrix [15,16,17,18] This method overcomes many limitations of free enzymes or conventional immobilization by letting the cells do the whole procedure [19]. Surfacedisplayed enzymes possess high tolerance or stability in harsh conditions, especially when proteins are embedded in the bacterial cell wall, and can be reused in several process cycles [18, 19]
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